Nuclear Magnetic Resonance (NMR) Spectroscopy involves placing a molecule to be analyzed in a powerful magnetic field and irradiating it with a strong radio signal. The nuclei of the various atoms will align themselves with the magnetic field until energized by the radio signal. They then absorb this energy and re-radiate (resonate) it at a frequency dependent on the type of nucleus and the chemical environment as largely determined by bonding of the nucleus. Moreover, resonances can be transmitted from one nucleus to another, either through bonds or through 3-D space, thus giving information about the environment of a particular nucleus and nuclei in its vicinity.
Not all isotopes of the same element are NMR active. For larger molecules, such as proteins, a sufficiently strong signal in NMR spectra requires enrichment with NMR active stable isotopes. The most commonly used stable isotopes for macromolecular NMR are 13C and 15N.
NMR has been successfully applied to determine the structures of over 2,500 proteins. The list of successful structures is dominated by relatively small (<40 kDa) soluble proteins that can be readily expressed in bacteria, which are capable of producing large amounts of soluble proteins uniformly labeled with the stable isotopes 13C and 15N. However, not all proteins are amenable to production and purification using bacterial expression systems. This is particularly true for mammalian membrane proteins which may undergo significant post-translational modifications including appropriate folding, cross-linking of inter- and intra-molecular chains through disulphide bridges, glycosylation, acylation, phosphorylation and other chemical modifications.
The limited success of E. coli for mammalian membrane protein expression is a major barrier to NMR experiments because E. coli are currently the most economical system for producing 13C and 15N labeled proteins. Bacterial media for stable isotope labeling is relatively inexpensive. E. coli can grow on 13C-glucose or (13C-glycerol) as the sole carbon source, and 15NH4Cl or 15(NH4)2SO4 as a nitrogen source. The estimated cost of 13C/15N bacterial media is about $1,500 to $3,000 per liter (Studier, F. W. 2005 Protein Expr. Purif. 41(1):207-234). Pichia Pastoris is a methylotrophic yeast currently used as an alternative to bacteria for isotope labeling. P. Pastoris can ferment 13C-methanol as the major carbon source, supplemented with 13C-glycerol during a brief initial batch phase (again, inexpensive 15NH4+ salts serve as the sole nitrogen source). Although the basic ingredients are similar, the nature of large-scale fermentation requires constant infusion of different components over several days. As such, the cost of uniform 13C/15N isotope labeling rises to an estimated $36,800 per liter of fermentation (Van den burg et al. 2001 J. Biomol. NMR 20(3):251-261). Expression in higher eukaryotic cells grown in culture (insect and mammalian) is also relatively expensive, given that no simple carbon/nitrogen source can be used. Isotope-labeled amino acids must be supplemented one-by-one into the complex media, resulting in costs of approximately $23,000 per liter for labeled insect cells.
Thus, there is a need in the art for additional methods for the economical incorporation of stable isotopes into proteins for NMR studies, particularly with regard to mammalian membrane proteins of interest such as G-Protein Coupled Receptors (GPCRs).